To quantify expression of a gene with semi-quantitative RT-PCR, the cDNA is synthesized and equalized based on an internal control gene such as 18S rRNA. The gene of interest is then amplified with a specific primer and PCR product is run on a gel and quantified. In some cases, PCR product(s) resulting from non-specific reactions may result, but these will be excluded when quantifying the gene expression.
My question is: if the same job is carried out with realtime RT-PCR, with it be able to descriminate accurately between products of specific reaction and other unintended products?
You contributions are greatly appreciated.
By performing melting curve analysis at the end of your qPCR run you can see if your primers are producing only one specific product (thus you have one melting temperature) or they generate several products in the reactions and thus they are not specific.
Best
Thanks Omar and Artur, but what if I do have a non specific reaction? How could I correct this in the data of real time PCR. I am working on a gene family with high sequence homology and it is hard to avoid non specific reactions in all genes.
Yes as Artur mentioned, primer design is the key. I was able to quantify two recently duplicated highly similar orthologs of gene AP3 by this. There was a difference of just 2 nucleotides in the 3' end of the primer binding the coding strand.
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